Probiotic bacteria have a strong potential in biomedicine owing to their ability to induce various beneficial health effects. Bacterial cell surface constituents play a key role in establishing tight interactions between probiotics and their host. Yet, little is known about the spatial organization and biophysical properties of the individual molecules. In this paper, we discuss how we have been using atomic force microscopy imaging and force spectroscopy to probe the nanoscale surface properties of Gram-positive lactic acid bacteria, with an emphasis on probiotic strains. Topographic imaging has enabled us to visualize bacterial cell surface structures (peptidoglycan, teichoic acids, pili, polysaccharides) under physiological conditions and with unprecedented resolution. In parallel, single-molecule force spectroscopy has been used to localize and force probe single cell surface constituents, providing novel insights into their spatial distribution and molecular elasticity.